Interaction of polymorphonuclear leukocytes with smooth and rough strains of Brucella abortus.

The bactericidal activity of guinea pig and human polymorphonuclear leukocytes (PMNs) against a smooth-intermediate strain (45/0) and a rough strain (45/20) of Brucella abortus has been examined. After incubation for 120 min, guinea pig PMNs incubated with either the smooth strain 45/0 or the rough strain 45/20 exhibited no bactericidal activity against the former and caused only a 34% decrease in viability of the latter. Human PMNs were more bactericidal than guinea pig PMNs to both strains; however, the killing of strain 45/20 by human PMNs was less than that observed in control experiments with S. aureus strain 502A. Both strains of B. abortus readily associated with guinea pig and human PMNs, and the bacteria were apparently ingested without stimulation of the hexose monophosphate pathway. Lysates (10 micrograms/ml, pH 5.5), prepared from guinea pig or human granules, were not particularly toxic to either strain unless supplemented with H2O2 and a halide (I- or Cl-). An oxygen-dependent killing system appeared to be lethal against both strains of B. abortus, with I- being more active than Cl- in the presence of H2O2 and granule lysate. The data suggest that degranulation after ingestion of Brucella by phagocytes does not occur due to the lack of a proper stimulus or possibly the baccilli actively inhibit the degranulation process thereby protecting the microbe from killing systems normally effective against extracellular parasites.     

Vaccination with Brucella abortus rough mutant RB51 protects BALB/c mice against virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis.

Vaccination of BALB/c mice with live Brucella abortus RB51, a stable rough mutant, produced protection against challenge with virulent strains of Brucella abortus, Brucella melitensis, and Brucella ovis. Passive-transfer experiments indicated that vaccinated mice were protected against B. abortus 2308 through cell-mediated immunity, against B. ovis PA through humoral immunity, and against B. melitensis 16M through both forms of immunity. Live bacteria were required for the induction of protective cell-mediated immunity; vaccination with whole killed cells of strain RB51 failed to protect mice against B. abortus 2308 despite development of good delayed-type hypersensitivity reactions. Protective antibodies against the heterologous species were generated in vaccinated mice primarily through anamnestic responses following challenge infections. Growth of the antigenically unrelated bacterium Listeria monocytogenes in the spleens of vaccinated mice indicated that nonspecific killing by residual activated macrophages contributed minimally to protection. These results encourage the continued investigation of strain RB51 as an alternative vaccine against heterologous Brucella species. However, its usefulness against B. ovis would be limited if, as suggested here, epitopes critical for protective cell-mediated immunity are not shared between B. abortus and B. ovis.     

High susceptibility of human dendritic cells to invasion by the intracellular pathogens Brucella suis, B. abortus, and B. melitensis

Bacteria from the Brucella genus are able to survive and proliferate within macrophages. Because they are phylogenetically closely related to macrophages, myeloid dendritic cells (DCs) constitute potential targets for Brucella bacteria. Here we report that DCs display a great susceptibility to Brucella infection. Therefore, DCs might serve as a reservoir and be important for the development of Brucella bacteria within their host.     

Brucellacidal activity of human and bovine polymorphonuclear leukocyte granule extracts against smooth and rough strains of Brucella abortus.

The microbicidal activities of freeze-thaw and high-salt extracts of human and bovine polymorphonuclear leukocyte (PMN) granules were tested against a smooth intermediate strain (45/0) and a rough strain (45/20) of Brucella abortus which differ in virulence and survival within PMNs. Freeze-thaw extracts of human PMN granules were more brucellacidal than high-salt extracts when supplemented with hydrogen peroxide (H2O2) and potassium iodide (KI), whereas the opposite was found with freeze-thaw and high-salt extracts of bovine PMN granules. There was no oxygen-independent killing of either the smooth or rough strain of B. abortus by amounts of granule extracts which caused 100% killing of a deep rough mutant (Re) of Salmonella typhimurium. The oxygen-dependent brucellacidal activity of granule extracts was dependent on concentrations of myeloperoxidase (MPO) units, H2O2, and KI. Maximal brucellacidal activity was observed at pH 5.5 to 6.0. The smooth strain, 45/0, was more resistant to oxygen-dependent killing by granule extracts than was the rough strain, 45/20. Granule extracts were more brucellacidal than purified MPO at equivalent levels of MPO enzyme units, suggesting that at least one other reaction enhances killing by the MPO-H2O2-I- system.     

Mitogenic activity of cell wall components from smooth and rough strains of Brucella abortus.

On the basis of [3H]thymidine incorporation by normal mouse spleen cell cultures, cell wall preparations from a smooth (45/0) strain and a rough (45/20) strain of Brucella abortus were strongly mitogenic. On the other hand, none of several subcomponents extracted from the cell wall preparations, including aqueous and phenolic lipopolysaccharides, was active. These results contrast with the marked mitogenic activity of lipopolysaccharides isolated from other gram-negative bacteria such as Salmonella typhimurium.     

Chemical characterization and biological properties of lipopolysaccharides isolated from smooth and rough strains of Brucella abortus.

The chemical composition and some of the biological activities of lipopolysaccharides (LPS) extracted from a smooth-intermediate strain (45/0) and a rough strain (45/20) of Brucella abortus have been examined. LPS were found in both the phenolic and aqueous extraction phases of strain 45/0, but only in the aqueous phase of 45/20. The phenolic LPS contained 9- to 16-fold-lower levels of heptose and reduced amounts of dideoxyaldoses compared with aqueous fractions. The major neutral sugars were glucose, galactose, and mannose. beta-Hydroxymyristic-acid, a common marker of enteric LPS, was not detected. Fatty acids present in highest amounts were hydroxylated and nonhydroxylated species with chain lengths of 16, 18, and 20 carbons. Only the phenolic LPS of strain 45/0 exhibited mouse lethality and a curable wasting disease; however, both phenolic and aqueous fractions caused carbohydrate depletion in mice. The toxicity of aqueous LPS could not be potentiated with Pb(OAc)4. These data, coupled with the lack of mitogenic activity for B-lymphocytes, are indicative of the unique structure-function relationships of Brucella LPS.     

迁移性树突状细胞与淋巴结内树突状细胞在抗B.suis免疫应答中的作用

目的:研究迁移性树突状细胞与淋巴结内树突状细胞在抗猪布鲁氏菌免疫应答中的作用。方法:48只BALB/c小鼠随机分为3组:Ⅰ组为正常对照组,阴道滴注PBS;Ⅱ组为巨噬细胞(Mφ)清除组,每只小鼠阴道滴入清除剂;Ⅲ组为未清除Mφ组,每只小鼠阴道滴注同体积的liposome-PBS;48小时后Ⅱ组、Ⅲ组均阴道接种猪布鲁氏菌,对照组阴道滴注PBS。各组小鼠分别在接种12、24、48和72小时采集血液和髂内淋巴结,采用免疫组织化学染色法观察淋巴结内树突状细胞(DC)的分布,用ELISA法检测血清中IFN-γ和IL-4的含量。结果:未清除Mφ组小鼠接种布鲁氏菌后髂内淋巴结内有大量DC迁移,至12小时血清IFN-γ水平显著增高,与对照组相比差异显著(P0.05),48小时达到高峰,显著高于对照组(P0.01)。清除Mφ组小鼠接种布鲁氏菌后,髂内淋巴结内没有明显的DC迁移,各时间点血清IFN-γ水平明显高于PBS对照组(P0.05),但明显低于未清除Mφ组(P0.05)。各组IL-4水平的差异无统计学意义(P0.05)。结论:迁移性DC和淋巴结内DC在启动抗布鲁氏菌细胞免疫应答中均发挥重要作用,并具有协同作用的特点。     

Brucella abortus rough mutants are cytopathic for macrophages in culture

Rough mutants of Brucella spp. are attenuated for survival in animal models. However, conflicting in vitro evidence has been obtained concerning the intracellular survival of rough mutants. Transposon-derived rough mutants isolated in our laboratory were previously shown to exhibit small but significant reductions in intracellular survival in a 12-h in vitro assay. Several recent publications report that rough mutants exhibited increased macrophage uptake relative to their smooth parental strains, and a reduction in numbers at the end of the assay has been interpreted as intracellular killing. In an effort to explore the role of O antigen in the interaction between Brucella abortus and macrophages, we have monitored the uptake of rough mutants and survival in vitro by using the murine macrophage cell line J774.A1. The results confirm a 10- to 20-fold-increased uptake of rough mutants over that of smooth organisms under standard conditions. Recovery of the rough mutants persisted up to 8 h postinfection, but at the point when intracellular replication of the smooth organisms was observed, the number of rough organisms recovered declined. Fluorescence microscopy revealed the intracellular multiplication of both smooth and rough organisms, and assays performed in the absence of antibiotic confirmed the replication of the rough organisms. Examination by phase-contrast microscopy revealed the lytic death of macrophages infected with the rough mutants, which was confirmed by the release of lactate dehydrogenase (LDH) from the cell cytoplasm. Thus, the decline in the number of rough organisms was the result of the lysis of macrophages and not from intracellular killing. The cytopathic effect is characterized as necrotic rather than apoptotic cell death based on early LDH release, annexin V and propidium iodide staining, morphological changes of infected cells and nuclei, and glycine protection. The cytopathic effect was observed with macrophages at multiplicities of infection (MOIs) of as low as 20 and was not observed with epithelial cells at MOIs of as high as 2000. These findings suggest a role for O antigen during the early stages of host-agent interaction that is essential in establishing an intracellular niche that maintains and supports persistent intracellular infection resulting in disease.     

Interaction of Neisseria meningitidis with human dendritic cells

Infection with Neisseria meningitidis serogroup B is responsible for fatal septicemia and meningococcal meningitis. The severity of disease directly correlates with the production of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, and IL-8. However, the source of these cytokines has not been clearly defined yet. Since bacterial infection involves the activation of dendritic cells (DCs), we analyzed the interaction of N. meningitidis with monocyte-derived DCs. Using N. meningitidis serogroup B wild-type and unencapsulated bacteria, we found that capsule expression significantly impaired neisserial adherence to DCs. In addition, phagocytic killing of the bacteria in the phagosome is reduced by at least 10- to 100-fold. However, all strains induced strong secretion of proinflammatory cytokines TNF-alpha, IL-6, and IL-8 by DCs (at least 1,000-fold at 20 h postinfection [p.i.]), with significantly increased cytokine levels being measurable by as early as 6 h p.i. Levels of IL-1beta, in contrast, were increased only 200- to 400-fold at 20 h p.i. with barely measurable induction at 6 h p.i. Moreover, comparable amounts of cytokines were induced by bacterium-free supernatants of Neisseria cultures containing neisserial lipooligosaccharide as the main factor. Our data suggest that activated DCs may be a significant source of high levels of proinflammatory cytokines in neisserial infection and thereby may contribute to the pathology of meningococcal disease.     

Interaction of Mycobacterium avium with human monocyte-derived dendritic cells

The mechanism by which mycobacteria elicit class I-restricted T-cell responses remains undefined because these organisms have been shown to reside exclusively within membrane-bound vesicles in macrophages (Mphi), their primary host cells. We studied the interaction of M. avium with dendritic cells (DC) because they are the most potent antigen-presenting cells and are abundant at M. avium infection sites. We observed that both DC and Mphi, generated from human peripheral blood monocytes by short-term culture, internalized M. avium. The onset of programmed cell death and the percentage of apoptotic cells in infected DC and Mphi were comparable. However, following infection, DC secreted significantly larger amounts of interleukin-12, but not interleukin-1beta, than infected autologous Mphi. Further analysis of infected cells showed that while phagosomes failed to acidify in both M. avium-infected DC and Mphi, bacilli grew more slowly in DC. Electron microscopy studies revealed that M. avium resided within endocytic vacuoles in both cell types. The vacuolar membrane surrounding some bacilli in approximately 10% of the vacuoles in DC possessed several breaks. The importance of this finding will have to be addressed in future studies.     

负载布氏菌WboA-/-S2株的树突状细胞对淋巴结T细胞的活化作用

目的:研究猪种布氏菌WboA-/-S2株对骨髓源性树突状细胞(BMDC)的生物学特性及启动T细胞应答能力的影响。方法:用粗糙型布氏菌WboA-/-S2株负载BALB/c小鼠的BMDC,并以光滑型S2株作为对照,采用瑞氏-姬姆萨染色观察细胞形态学变化并计算吞噬率;用固体培养基涂布法测定负载后不同时间点细胞的载菌量;用ELISA检测负载后不同时间点BMDC分泌IL-12和TNF-α的量以及与淋巴结T细胞共培养上清中IFN-γ和IL-4的含量。结果:BMDC对WboA-/-S2株的吞噬率高于对S2株的吞噬率(P<0.05),负载1小时BMDC的载菌量明显高于负载S2株的载菌量(P<0.05),但在24小时,WboA-/-S2株负载BMDC的载菌量明显低于S2株负载组(P<0.05)。WboA-/-S2株负载BMDC不同时间点上清中IL-12和TNF-α含量明显高于S2株负载组(P<0.05)。且负载WboA-/-S2株的BMDC刺激T细胞所产生的IFN-γ量均明显高于S2株(P<0.05)。结论:WboA-/-S2株较S2株更易被BMDC吞噬和杀死,其活化BMDC、诱导T细胞应答的能力也明显强于S2株。     

Surface macromolecules and virulence in intracellular parasitism: comparison of cell envelope components of smooth and rough strains of Brucella abortus.

The surface topography of whole cells and the chemical composition of cell envelopes of a smooth-intermediate strain (45/0) and a rough strain (45/20) of Brucella abortus was examined. Electron microscopy of whole cells and thin sections did not reveal any gross surface difference(s). Only minor quantitative differences were observed in total lipids, proteins, and the murein layer. However, the lipopolysaccharide composition of the two strains was quite different. Both phenol- and water-soluble lipopolysaccharide fractions were obtained from the strain of higher virulence (45/0), whereas only aqueous lipopolysaccharide could be isolated from the rough strain. In addition to being toxic, the phenol-soluble lipopolysaccharide may be a key virulence factor in intracellular survival of B. obortus within phagocytic cells.     

Growth of Brucella abortus in macrophages from resistant and susceptible mouse strains

C57Bl/10 mice have a superior ability to control chronic infections with virulent strains of the intracellular bacteria Brucella abortus compared with BALB/c mice. While a number of differences in the cytokines produced by lymphocytes following infection of these two strains of mice have been shown, macrophages have not been evaluated for their role in conveying relative resistance. The importance of macrophages in control of brucella infections is demonstrated by the observations that intracellular survival of various strains of B. abortus directly correlates with their virulence in vivo, and the ability of macrophages to control brucellae in vitro has been shown to correlate with a brucella-resistant phenotype in ruminants. While both BALB/c and C57Bl are Nramp-susceptible mouse strains, additional differences in macrophage function outside of the Nramp1 gene effects could influence susceptibility to brucellosis. The studies conducted here comparing the ability of macrophages from C57Bl/10 and BALB/c mice indicate that the macrophages from resistant mice did not control intracellular growth of B. abortus strain 2308 more efficiently than those from the susceptible mice, either in the absence of, or following, interferon-gamma activation or iron supplementation. A number of different conditions for culturing macrophages were evaluated to rule out the influence of antibiotics on the conclusions drawn from the results.     

Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR.

Several PCR assays which identify the genus Brucella but do not discriminate among species have been reported. We describe a PCR assay that comprises five oligonucleotide primers which can identify selected biovars of four species of Brucella. Individual biovars within a species are not differentiated. The assay can identify three biovars (1, 2, and 4) of B. abortus, all three biovars of B. melitensis, biovar 1 of B. suis, and all B. ovis biovars. These biovars include all of the Brucella species typically isolated from cattle in the United States, a goal of the present research. The assay exploits the polymorphism arising from species-specific localization of the genetic element IS711 in the Brucella chromosome. Identity is determined by the size(s) of the product(s) amplified from primers hybridizing at various distances from the element. The performance of the assay with U.S. field isolates was highly effective. When 107 field isolates were screened by the described method, there was 100% agreement with the identifications made by conventional methods. Six closely related bacteria (Agrobacterium radiobacter, Agrobacterium rhizogenes, Ochrobactrum anthropi, Rhizobium leguminosarum, Rhizobium meliloti, and Rhodospirillum rubrum) and two control bacteria (Bordetella bronchiseptica and Escherichia coli) tested negative by the assay.     

Contradictory Roles for Antibody and Complement in the Interaction of Brucella abortus with Its Host

Abstract

The ability of serum complement to kill bacteria has been linked to host resistance to Gram-negative bacteria. A mechanism for killing extracellular organisms during early invasion, following release from infected phagocytic cells, or during bacteremia would contribute to a host's ability to resist disease. In fact, the ability of serum complement to kill bacteria has been linked to disease resistance. Brucella abortus are Gram-negative intracellular pathogens. Resistance to these bacteria involves the coordinated activities of the cellular and humoral immune systems. The existence of serum-resistant forms of B. abortus has been established, and it has been shown that these bacteria can resist the killing action of complement even in the presence of specific antibody. Antibody is usually necessary for complement-mediated killing of smooth (virulent) forms of Gram-negative bacteria. An anomolous situation exists with some isolates of smooth B. abortus. Sera containing high titers of specific antibody do not support killing unless they are diluted. In the bovine, this phenomenon is associated with IgGl and IgG2 antibodies. This finding may account for the lack of positive correlation between antibody levels and resistance to disease, which has led, perhaps wrongly, to the idea that antibody and complement are not important in resistance to brucellosis.

Available evidence suggests that antibody may have contradictory roles in the interactions between a host and bacteria. Avirulent (rough) forms of the organism would be rapidly killed by complement shortly after invasion, but serum-resistant smooth forms would survive and invade resident phagocytic cells. During the process of invasion and phagocytosis, the bacteria would initiate an immune response. With time, some B. abortus organisms would be released from infected phagocytic cells. In the early stages of this process, the bacteria would encounter IgM antibody and low concentrations of IgG antibody. These would cause complement-mediated killing, and infection would be restricted to resident phagocytic cells. However, the immune response to B. abortus antigens would be intensified, and IgG antibody levels would increase. High concentrations of antibody do not support complement-mediated killing of extracellular B. abortus, but the bacteria would be opsonized by antibody and complement component fragments. This would lead to increased phagocytosis of extracellular B. abortus as they appear, and concomitant extension of disease. Because die high levels of antibody would block complement-mediated lulling of B. abortus, resistance to disease at this point would be dependent on cell-mediated immunity.     

Brucella suis prevents human dendritic cell maturation and antigen presentation through regulation of tumor necrosis factor alpha secretion

Brucella is a facultative intracellular pathogen and the etiological agent of brucellosis. In some cases, human brucellosis results in a persistent infection that may reactivate years after the initial exposure. The mechanisms by which the parasite evades clearance by the immune response to chronically infect its host are unknown. We recently demonstrated that dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to Brucella infection and are a preferential niche for the development of the bacteria. Here, we report that in contrast to several intracellular bacteria, Brucella prevented the infected DCs from engaging in their maturation process and impaired their capacities to present antigen to naïve T cells and to secrete interleukin-12. Moreover, Brucella-infected DCs failed to release tumor necrosis factor alpha (TNF-α), a defect involving the bacterial protein Omp25. Exogenous TNF-α addition to Brucella-infected DCs restored cell maturation and allowed them to present antigens. Two avirulent mutants of B. suis, B. suis bvrR and B. suis omp25 mutants, which do not express the Omp25 protein, triggered TNF-α production upon DC invasion. Cells infected with these mutants subsequently matured and acquired the ability to present antigens, two properties which were dramatically impaired by addition of anti-TNF-α antibodies. In light of these data, we propose a model in which virulent Brucella alters the maturation and functions of DCs through Omp25-dependent control of TNF-α production. This model defines a specific evasion strategy of the bacteria by which they can escape the immune response to chronically infect their host.Brucella is a facultative intracellular α2-proteobacterium that induces chronic infections in a wide variety of mammals, including field ruminants, humans, and marine mammals. In addition to the attention received from its classification as a potential weapon for bioterrorism (41), this bacterium is principally known because of its ability to induce infectious abortion in domestic animals and because brucellosis is the most frequent anthropozoonosis (20). The three most infectious species in humans are B. melitensis, B. abortus, and B. suis. Infection occurs through inhalation of aerosols or ingestion of infected food. Following invasion of the lymphoid system, the bacteria develop within mononuclear phagocytes, and infected cells play a crucial role in the dissemination of the bacteria in specific locations of the body. Also known as Malta fever, human brucellosis consists of an acute infection, characterized by undulant fever and asthenia, which evolves in 30% of infected patients into a chronic phase with erratic recurrent fevers and localized infections, such as endocarditis, encephalitis, and spondylitis. Chronic brucellosis patients display a T helper 2 (Th2)-specific immune response (19, 43). In mice, which are not natural hosts for Brucella and display a certain resistance to infection, the protection is conferred by a Th1-oriented immune response depending on gamma interferon-producing CD4⁺ T lymphocytes (2, 3, 46). Therefore, the ability of Brucella to chronically infect human hosts seems to be related to its capacity to avoid establishment of a protective Th1-specific response (7, 19, 43, 55).For several years, our laboratory and others have studied the interaction of Brucella with macrophages and identified several virulence mechanisms implicated in the interaction of Brucella with the innate immune system (1, 9, 10, 18, 21, 25, 29, 30, 39). Nevertheless, due to reduced implication of macrophages in the initiation of a specific immune response, the macrophage infection model is not suitable when the adaptive immune response is considered in the context of the Brucella-host interaction. Myeloid dendritic cells (DCs) have naturally emerged as interesting models. DCs serve as sentinels for the immune system; they ingest pathogens at the site of infection and migrate to secondary lymphoid organs, where they present pathogen-derived antigens to naïve T lymphocytes, thus initiating the specific immune response. Pathogens could target DCs during early stages of infection as a way of disabling and evading host immune responses. In a previous publication, we showed for the first time that human DCs are highly permissive cellular hosts for Brucella (5). In this report, we analyze the consequences of Brucella infection for DC physiology, particularly for maturation processes and antigen presentation to naïve T cells. Relationships with some bacterial proteins were studied, and the importance of DC infection for Brucella''s virulence strategy and the pathogenesis of human brucellosis is discussed.     

Modulation of immune response by killed Brucella abortus organisms: comparison of the effects of smooth and rough strains on T-dependent responses.

Inactivated Brucella abortus organisms of the smooth (S) or rough (R) strain were tested comparatively on two T-dependent immune responses: mixed-lymphocyte reaction and delayed-type hypersensitivity. The intravenous injection of S organisms depressed the two tests, whereas R organisms increased mixed-lymphocyte reaction and did not alter delayed-type hypersensitivity significantly. This observation may be helpful in understanding the differences in adjuvant properties of S and R brucellae.     

Plaque-forming cells in mice after experimental infection with Brucella abortus.

Cells producing antibody to brucella lipopolysaccharide were detected in spleens of mice infected with Brucella abortus 19 by a hemolytic plaque assay. The appearance of immunoglobulin M-producing cells preceded humoral antibodies. The primary plaques were observed 5 days after inoculation, and they were still present by day 70.